Antimicrobial resistance is an emerging global health issue. Data on the epidemiology of multidrug-resistant organisms are scarce for Africa, especially in HIV-infected individuals who often have frequent contact with healthcare. We investigated the prevalence of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) carriage in stool among HIV-infected children attending an HIV outpatient department in Harare, Zimbabwe.We recruited children who were stable on antiretroviral therapy (ART) attending a HIV clinic from August 2014 to June 2015. Information was collected on antibiotic use and hospitalization. Stool was tested for ESBL-E through combination disc diffusion. API20E identification and antimicrobial susceptibility was performed on the positive samples followed by whole genome sequencing.Stool was collected from 175/202 (86.6 %) children. Median age was 11 [inter-quartile range (IQR) 9–12] years. Median time on ART was 4.6 years (IQR 2.4–6.4). ESBL-Es were found in 24/175 samples (13.7 %); 50 % of all ESBL-Es were resistant to amoxicillin-clavulanate, 100 % to co-trimoxazole, 45.8 % to chloramphenicol, 91.6 % to ceftriaxone, 20.8 % to gentamicin and 62.5 % to ciprofloxacin. ESBL-Es variously encoded CTX-M, OXA, TEM and SHV enzymes. The odds of ESBL-E carriage were 8.5 times (95 % CI 2.2–32.3) higher in those on ART for less than one year (versus longer) and 8.5 times (95 % CI 1.1–32.3) higher in those recently hospitalized for a chest infection.We found a 13.7 % prevalence of ESBL-E carriage in a population where ESBL-E carriage has not been described previously. Antimicrobial resistance (AMR) in Africa merits further study, particularly given the high HIV prevalence and limited diagnostic and therapeutic options available. The widespread use of antibiotics worldwide has resulted in the emergence of a global problem of antimicrobial resistance (AMR). Over the past decade, there has been an increasing prevalence of carriage of and infection with multidrug-resistant organisms (MDROs) such as extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) and carbapenemase-producing Enterobacteriaceae (CPE) . ESBL anThe emerging global threat of antimicrobial resistance (AMR) is particularly concerning for developing countries, as infections with ESBL-E and CPE are difficult to treat due to limited antibiotic treatment options that are not readily available in most resource-limited settings.Escherichia coli isolated pertained to the pandemic ST131 group [While the epidemiology, phenotypic and genotypic characteristics of MDROs are well described in Europe, North America and Asia , such da31 group .Sub-Saharan Africa is the epicentre of the HIV epidemic, hosting an estimated 70 % (approximately 25 million) of the world’s HIV-infected population in 2014 . IndividThe aim of our study was to determine the prevalence of ESBL-E and CPE carriage in stool among HIV-infected children accessing outpatient HIV care in Harare, Zimbabwe.This study was conducted at the paediatric HIV care clinic at Harare Central Hospital, the largest public sector hospital in Harare, Zimbabwe. The clinic provides HIV care to over 3000 children. This study was nested in a larger study investigating the epidemiology of HIV-associated chronic lung disease (INHALE study). Perinatally HIV-infected children aged between 6 to 16 years who had been taking antiretroviral therapy (ART) for at least 6 months and were not acutely unwell were recruited consecutively over a 10-month period, with guardian consent and participant assent. Ethical approval for the study and shipment of specimens was granted by the Medical Research Council of Zimbabwe and the Research Council of Zimbabwe.A questionnaire recording socio-demographic details and clinical history, including duration of ART use and previous hospitalization, was administered on site to all participants/guardians. CD4 count was measured using the Alere PIMA analyser  and HIV viral load was measured using the COBAS Ampliprep/Taqman 48 version 2.0 . Participants were asked to provide a fresh stool sample on site or at home on the morning of the second study visit (INHALE study), using a stool collection kit and transported on ice if the participant had a freezer.E. coli), any AmpC genes would be likely to be plasmid-associated.Freshly collected stool samples were inoculated onto cysteine lactose electrolyte-deficient (CLED) medium with the addition of a cefpodoxime disc  for ESBL-E screening, and an ertapenem disc for CPE screening. Each colonial morphotype of oxidase-negative Gram-negative isolates that grew within a diameter zone of inhibition of ≤20 mm around the cefpodoxime disc or ≤28 mm around the ertapenem disc was subcultured for ESBL-E and CPE screening, respectively, and based on British Society for Antimicrobial Chemotherapy (BSAC) breakpoints . ESBL-E Preliminary identification of these resistant organisms was performed using API20E  and was subsequently confirmed using MALDI-TOF . Antimicrobial susceptibility testing (AST) was performed by disc diffusion testing following EUCAST methodology . Isolatek-mer values from 47 to 71. blast searching against multi-locus sequence type (MLST) databases was used to identify the nearest match for each allele, followed by assignation of allelic profiles.DNA was extracted from RNase-treated lysates using a Qiasymphony DSP DNA midi kit . DNA libraries were prepared using the NexteraXT sample preparation method and sequenced with a standard 2×101 base protocol on a HiSeq 2500 Instrument . Sequence data from the sequencing runs were deplexed and trimmed, and the presence of antimicrobial resistance genes was determined with ‘Genefinder’ as described previously . Genome 2 test or Fisher’s exact (or non-parametric Mann–Whitney) test and univariate logistic regression were used to assess the association between the variables of interest and the occurrence of ESBL-E. A two-tailed P-value of <0.05 was considered to represent a statistically significant association. All the variables were considered regardless of their initial P-value into a multivariate logistic regression to assess the adjusted net effect of the independent variables.Data were entered using Cardiff TELEFORM Intelligent Character Optical Mark Recognition Software (version 10.7) and analysed using STATA Version 13.1 . Descriptive statistics were performed on the demographic and baseline characteristics of the participants. For continuous variables, non-parametric medians and inter-quartile ranges (IQRs) were calculated and for categorical variables, frequencies and percentages were determined. A χ−1, and 67.0 % of the participants had a viral load of <50 copies ml−1. Only 2 % of the participants had been admitted to the hospital for a chest infection/pneumonia in the previous 12 months.A total of 202 eligible HIV-infected participants were recruited between August 2014 and June 2015, of whom 175/202 (86.6 %) were able to provide a stool sample and were included in this study. The median age was 11 (IQR 9–12) years and 48 % were female. The median time on ART was 4.6 years (IQR 2.4–6.4)  and 96 %E. coli and 1/24 (4.2 %) as K. pneumoniae. The other four resistant isolates included three isolates identified as E. coli (not ESBL-producing) and one isolate identified as Acinetobacter baumannii, which was not further investigated. MALDI-TOF testing confirmed the identification of all organisms (data not shown). No CPE were detected.Laboratory analysis of the stool samples showed that 28/175 (16.0 %) were positive for possible ESBL-Es on initial screening. On confirmatory testing, 24 samples did contain an ESBL-E giving an estimated ESBL-E prevalence of 13.7 % (95 % CI 8.9–19.7) in this population. Of these, 23/24 (95.8 %) were identified as P=0.001) whereby participants on ART for less than 1 year had 7.7 times (95 % CI : 2.03–29.0) the odds of carrying an ESBL-E (P=0.033). These factors remained significant when considered in the final multivariate model when participants had eight times the odds of carrying ESBL-Es. Demographic and other clinical factors were included in the multivariate model despite P-values>0.2, and no significant association was observed even in the adjusted model.Bivariate analysis to assess the factors associated with ESBL-E carriage found no demographic association with ESBL-E carriage. Independent association, however, was found between ESBL-E carriage and duration on ART  were genetically diverse, represented by 16 different MLSTs . Molecular characteristics of the isolates relevant to this paper are displayed in E. coli were predominated by nine ST131 isolates with the remainder comprised of two isolates each of STs 48, 656 and 1589, and 11 singletons. The ESBLs were predominated by CTX-M enzymes, comprising mainly group-9 CTX-Ms (six CTX-M-27 and four CTX-M-14) followed by seven CTX-M-15 and four CTX-M-3, both in group 1 (K. pneumoniae).The 27 isolates (i.e. 23 ESBL-positive  group 1 . Three iP=0.001), as was having been admitted to hospital for a chest infection in the past 12 months  reported outpatient setting carriage prevalence estimate of 59 % (79/134) in rectal swabs from healthy young children in the Central African Republic and higher than the 7.4 % in nasopharygeal swabs from HIV-infected children in Cape Town. Among the ESBL-E isolates, there was a high level of resistance to co-trimoxazole, ciprofloxacin, chloramphenicol and gentamicin. Participants enrolled in the current study were often repeatedly exposed to inpatient and outpatient environments and to have received multiple courses of antibiotics throughout their short lives due to their chronic immunodeficient state. Carriage was associated with having started ART recently and having been hospitalized with a chest infection in the previous 12 months. Unfortunately, reliable information on previous antibiotic usage was not available and parents were often unsure of the exact treatment their child had received.Two recent reviews on MDRO prevalence in Africa reported ESBL-E prevalence rates ranging from 1.3–22.8 % in urine and 0.7–75.8 % in blood . UnfortuP=0.033). This points to the possibility of having acquired the ESBL-E while in hospital. The other potential significant risk factor was having been on ART for less than 1 year (P=0.002). These children are likely to have been immunosuppressed more recently and to have received antibiotics and/or to have recent contact with healthcare services, compared to children well established on ART for years.Being admitted to hospital within the previous 12 months for a chest infection was associated with ESBL-E carriage (The ESBL-Es identified in this study were resistant to multiple classes of antibiotics. All isolates were resistant to co-trimoxazole with less resistance to ciprofloxacin (60 %), chloramphenicol (45 %), gentamicin 20 %) and temocillin (12 %), and none to amikacin. The level of resistance reflects the usage of these antibiotics in the public sector in Zimbabwe. All HIV-infected individuals receive long-term co-trimoxazole for prophylaxis, and WHO guidelines recommend continuation of co-trimoxazole in children even following immune reconstitution with ART . Ciprofl % and teE. coli aligns with the predominance of this ST in many other parts of the world [E. coli we observed, including the ST131 isolates, encoded ESBLs alongside multiple other resistances . Concurrent antimicrobial resistance limits treatment options and is known to be typically encoded on plasmids and transposons that are capable of intra- and inter-species spread. If carriage in these children were to result in urinary or bloodstream infections, gentamicin would be the only available antibiotic with a reasonable chance of treatment success.Our observation of multiple cases affected by ST131 he world  and its The study was conducted in the public sector among asymptomatic and previously unstudied HIV-infected children and adolescents. Multiple standardized and well-established methods were used to identify ESBL-E phenotypically and genotypically. However, the sample size was small resulting in reduced power to identify risk factors. A limitation of this study was that no comparison was made with HIV-negative children to ascertain whether ESBL-E carriage was associated with HIV status. Thus whether the prevalence of 13.7 % is specific to this patient population or due to a high carriage rate in the community is unknown and requires further investigation. Variables such as self-reported hospitalization are subject to recall bias, although we attempted to confirm contact with health services from patient-held clinical records. Participants may have been hospitalized for other infections, but as this study was embedded in a study investigating chronic lung disease, only hospitalisations for lung-related infections were recorded. However, respiratory tract infection is the most common infection among HIV-infected children, including among those who are immunocompetent.The ESBL-E prevalence identified in this study carries several important implications. First, the carriers themselves are at risk of becoming infected. Gram-negative sepsis due to ESBL-Es remains largely untreated in this setting and is associated with very high mortality. Second, these children are in recurrent contact with health services and, as such, the ESBL-E carriage has implications for infection control. Finally, this study is a reminder of how the use of ceftriaxone, a broad-spectrum third-generation cephalosporin widely used as a first-line antibiotic for sepsis and meningitis throughout Sub-Saharan Africa , might f


The expression and prognostic significance of the c-erbB-2 oncogene product was studied in 55 cases of childhood medulloblastoma. Forty-six of the 55 tumours (83.6%) expressed the c-erbB-2 product. The percentage of tumour cells expressing the c-erbB-2 product proved to be a significant indicator of patient outcome when analysed as both a categorical and a continuous variable. As a categorical variable, patients with more than 50% positive tumour cells had a significantly worse survival, with only 10% alive at 10 years vs 48% for those with less than 50% positive tumour cells (log rank P = 0.0049). To demonstrate that this observed prognostic significance was both independent and not a result of 'data-driven' categorisation, it was also entered into the Cox model as a continuous variable. Prognostic significance was retained in P = 0.038.


In the VDS-P arm, cisplatin 80 mg m−2 was administered on day 1, and vindesine 3 mg m−2 was administered on days 1, 8 and 15. In the CPT arm, CPT-11 100 mg m−2 was administered on days 1, 8 and 15. The median survival time was 50.0 weeks for patients on CPT-P, 45.6 weeks for those on VDS-P and 46.0 weeks for those on CPT , and the hazard ratio was 0.85 : 0.65–1.11) for CPT-P vs VDS-P and 0.83 (0.64–1.09) for CPT vs VDS-P. The response rate was 43.7% for patients on CPT-P, 31.7% for those on VDS-P and 20.5% for those on CPT. Major adverse reactions were grade 4 neutropenia observed in 37, 54 and 8% of the patients on CPT-P, VDS-P and CPT, respectively; and grades 3 and 4 diarrhoea observed in 12, 3 and 15% of the patients, respectively. CPT-P therapy produces comparable survival to VDS-P in patients with advanced NSCLC. CPT-11 monotherapy is not inferior to VDS-P in terms of survival. The CPT-11-containing regimen is one of the most efficacious and well tolerated in the treatment of advanced NSCLC.To determine a standard combination chemotherapy for patients with advanced non-small-cell lung cancer (NSCLC), we conducted a phase III trial of irinotecan (CPT-11) to test the hypotheses that CPT-11+cisplatin is superior to cisplatin+vindesine and that CPT-11 monotherapy is not inferior to cisplatin+vindesine. A total of 398 patients with previously untreated NSCLC were randomised to receive cisplatin+CPT-11 (CPT-P), cisplatin+vindesine (VDS-P) or CPT-11 alone (CPT). In the CPT-P arm, CPT-11 60 mg m In Japan, lung cancer is the leading cause of death among all the cancers, accounting for approximately 50 000 deaths annually . Non-smaCamptotheca acuminata. Differing from conventional antitumour drugs in mechanism of action, CPT-11 produces its effect by inhibiting the synthesis of DNA and RNA through inhibition of DNA topoisomerase I (Irinotecan hydrochloride (CPT-11) is a water-soluble derivative of camptothecin, an alkaloid originally extracted from the Chinese tree −2 week−1   Clinical Trials Group undertook a randomised study involving patients with NSCLC to determine whether there was a survival advantage for those patients treated with combination chemotherapy (vindesine (VDS)+CDDP or cyclophosphamide+doxorubicin+CDDP) over best supportive care (BSC). The results of that trial showed that the patients receiving VDS+CDDP had a statistically significant survival advantage over patients receiving BSC , liver , kidneys  and lungs . Patients with other concurrent malignancies or a history of other malignancies, active infection, diarrhoea (watery stool), paralytic ileus, pulmonary fibrosis, pericardial effusion, considerable pleural effusion or ascites, uncontrolled diabetes mellitus or symptomatic metastasis to the brain, were excluded. Informed consent was obtained from each patient before enrollment. Each institutional review board for human experimentation approved the protocol of this study.Patients with histologically or cytologically confirmed and previously untreated NSCLC were enrolled into this trial. Patients with stage IIIB or IV cancer were eligible if they had measurable disease and a life expectancy of at least 3 months. Additional inclusion criteria were age (15–75 years old); a performance status (PS) of 0–2 on the Eastern Cooperative Oncology Group (ECOG) scale; and adequate functional indices for bone marrow  using stage (IIIB/IV), PS (0–1/2) and institution as balancing variables  on days 1, 8 and 15 at a dose of 60 mg m3 receptor antagonist was given before CDDP administration. In the CPT-P arm, CDDP was administered after the CPT-11 infusion.CPT-11, diluted in ⩾500 ml of normal saline, was administered by i.v. infusion over 90 min. VDS was administered as an i.v. push with a running of 5–10 ml of normal saline. CDDP was administered i.v. as an undiluted solution and infused for a period of 60 min with more than 2600 ml of hydration and a diuretic before and after administration. To control CDDP-induced emesis, a 5-HT−3, platelet count was less than 100 000 mm−3, body temperature was 37.5°C or higher, or diarrhoea was grade 1  or higher on the ECOG scale.In all three arms, the dose of CPT-11 or VDS on day 8 or 15 was not given if the leucocyte count was less than 3000 mm−3 or more, platelet count had to be 100 000 mm−3 or more, serum creatinine concentration had to be normal, and diarrhoea and fever should have disappeared. If there was a delay greater than 4 weeks caused by persistent toxicity, patients in each arm were to be withdrawn from the study.In each arm, before the next course was started, the leucocyte count had to be 4000 mm−3, platelet count less than 50 000 mm−3 or diarrhoea had been grade 2  or higher on the ECOG scale, the dose of CPT-11 was reduced to 50 mg m−2 in the CPT-P arm, and to 80 mg m−2 in the CPT arm. If during the previous course the serum creatinine concentration was more than 1.5 times the upper normal limit, the dose of CDDP was reduced to 60 mg m−2 in the CPT-P arm and in the VDS-P arm.If during the previous course, the leucocyte count had been less than 1000 mmThis trial was independently monitored and performed according to GCP rules.The primary end point of this study was overall survival. Response rate, duration of response, time to disease progression and toxicity were all secondary end points.The stage of the disease was determined based on a complete medical history and physical examination, routine chest radiographs, fibreoptic bronchoscopy, computed tomography of the head, chest and abdomen and bone scintigraphy. The staging estimates were made according to the international staging system . Before Health-related quality of life (QOL) was assessed experimentally with a self-administered QOL questionnaire for cancer patients treated with anticancer drugs (QOL-ACD)  in one patient.From July 1995 to January 1998, 398 patients from 41 centres were entered into this study. Of the 398 patients randomised, 18 patients did not meet the eligibility criteria. The reasons for exclusion were: early stage (<IIIB) in five patients, previous treatment  in eight patients, other concurrent malignancies in two patients, age (>75 years) in one patient, presence of pericardial effusion in one patient, and leucocyte count (>12 000 mmThe patient characteristics at baseline are listed inThree patients received no treatment (one patient in the CPT-P arm and two patients in the CPT arm), two patients in the VDS-P arm received vincristine (VCR) instead of VDS, one patient in the CPT-P arm exceeded the daily dose of CPT-11 and CDDP, and one patient in the CPT-P arm received CDDP on days 1 and 8. These seven patients were included in the survival analysis, but excluded from response and toxicity analysis. In all, 109 of the 126 patients on CPT-P (87%), 97 of the 120 patients on VDS-P (81%) and 101 of the 127 patients on CPT (80%) received more than two courses of treatment, and 22 patients (18%) on CPT-P, 15 patients (13%) on VDS-P and 20 patients (16%) on CPT received more than four courses of treatment. The median number of courses administered per patient was 3 for CPT-P and 2 for both VDS-P and CPT. The number of treatment courses varied from one to six in each arm. In the CPT-P, VDS-P and CPT arms, 23, 10 and 16 patients were withdrawn from the study because of toxicities (including duplications); 28, 44 and 51 patients because of disease progression; 37, 27 and 19 patients due to patient's refusal; 10, 19, and 22 patients because of aggravated clinical symptom; 50, 32 and 28 patients due to investigator's medical decision; and 6, 9 and 9 patients because of other reasons, respectively. Most patients could repeat treatment courses every 28 days irrespective of the arm.−2 week−1) for both the CPT-P and VDS-P arms. The median dose intensity of CPT-11 was 30 mg m−2 week−1 (67% of planned dose) for the CPT-P arm and 61.3 mg m−2 week−1 (82% of planned dose) for the CPT arm. The median dose intensity of VDS was 1.5 mg m−2 week−1 (67% of planned dose).The number of treatment courses in which CPT-11 or VDS was administered on days 1, 8 and 15 totaled 164 courses  for the CPT-P arm, 66 courses  for the VDS-P arm and 182 courses  for the CPT arm. Dose omissions on day 8 and/or day 15 were because of diarrhoea and/or leucopenia in the CPT-P arm and the CPT arm, and because of leucopenia in the VDS-P arm. The median dose intensity of CDDP was the same  for CPT-P vs VDS-P and 0.83 (95% CI: 0.64–1.09) for CPT vs VDS-P.The survival curves for all eligible patients are shown in Figure 2P=0.838) for CPT-P vs VDS-P and 0.99  for CPT vs VDS-P.Subgroup analyses for survival were conducted using stage as one of the balancing variable factors  for CPT-P vs VDS-P and 0.70  for CPT vs VDS-P.Among patients with stage IV disease , the MSTThe Cox proportional hazards model was used to adjust for and determine the impact of prognostic factors and treatment on survival (χ2 test: P<0.001).Objective response data are listed in P=0.601). The median time to progression for the patients included in the efficacy analysis was 148 days for patients on CPT-P, 117 days for those on VDS-P and 100 days for those on CPT .The median duration of response for responders was 141 days for patients on CPT-P, 121 days for those on VDS-P, and 117 days for those on CPT . Grade 4 thrombocytopenia was more frequent in the CPT-P arm, but there were no thrombocytopenia-related complications.Major adverse reactions are listed in P=0.008). The regimens containing CDDP  were associated with a significantly higher occurrence of grade 3 nausea/vomiting as compared with the CPT alone regimen (P=0.001). Peripheral neurological symptoms were observed significantly more frequently in the VDS-P group than in the other two groups (P<0.001).Diarrhoea was the main nonhaematological sign of toxicity of the regimens containing CPT-11 (CPT-P and CPT); these two regimens were associated with a significantly higher occurrence of grades 3 and 4 diarrhoea as compared with the VDS-P regimen  monotherapy or DTX-containing regimens.Since 1990, newer agents such as CPT-11, paclitaxel (PTX), DTX, gemcitabine (GEM) and vinorelbine (VNB) have shown response rates of 20% or more when used alone in previously untreated NSCLC .Except for CPT-11, all four chemotherapeutic agents have been examined in many phase III trials in patients with advanced NSCLC and their roles have been mostly elucidated. No trial has been conducted, however, in order to establish the role of CPT-11 in advanced NSCLC. The present clinical trial was the first phase III study of CPT-11 in patients with advanced NSCLC. We planned this phase III trial in order to verify (1) whether CPT-P significantly prolongs the survival time and (2) whether CPT alone is not inferior in terms of the survival time, as compared with VDS-P, one of the most commonly used chemotherapeutic regimen that has been used in advanced NSCLC.vs an old platinum-based chemotherapy. Only the studies by The first results showed that CPT-P did not significantly prolong the survival time compared with VDS-P. Previous studies of PTX , similar to the present trial, the survival was also similar between VNB alone and VDS-P. Toxicity on CPT alone was mild except for slightly frequent diarrhoea. Based on these results, chemotherapy with the new single agent may be considered for patients who are unable to receive CDDP-containing regimens because it can produce the similar survival to conventional platinum-based combinations, and toxicity is relatively mild.The second results showed that CPT-11 alone was not inferior to VDS-P in terms of the survival time. In a study of P=0.004). CPT-11 alone also significantly prolonged the survival time in patients with stage IV NSCLC compared with VDS-P. Among stage IV patients, there was no bias in terms of background factors including PS , gender  and the rate of weight loss at baseline . There was no bias in the content of second-line treatment among the treatment arms. These are just the results of the subgroup analyses and their significance should be shown in another research.In the subgroup analyses according to the stage, in stage IV patients, the MST was 50.0 weeks on CPT-P and 36.4 weeks on VDS-P, with the CPT-P being superior to the VDS-P with respect to survival prolongation . The hazard ratio for CPT vs CPT-P was 1.03 (95% CI: 0.79–1.34).Comparing survival between CPT-P and CPT-11 alone, although this was not planned, no significant difference was shown between the two arms . This significant difference in the response rate did not lead to significant differences in the survival time. Similar results have been frequently reported in comparative studies of chemotherapeutic agents in advanced NSCLC. Various known and unknown prognostic factors were involved in this result. Of them, the major factor is that complete response (CR) is obtained only in small numbers of NSCLC patients receiving chemotherapy. In the present trial, the CR rate was just 2.4% (three out of 126) in the CPT-P arm, 0.8% (one out of 120) in the VDS-P arm and 0.8% (one out of 127) in the CPT arm.The overall response rate in this study was 43.7% on CPT-P, 31.7% on VDS-P and 20.5% on CPT  by Neutropenia and neurotoxicity were more frequent in the non-CPT-containing VDS-P arm, than in the CPT-containing CPT-P and CPT arms. Nausea/vomiting was more frequent in the CDDP-containing CPT-P and VDS-P arms, than in the non-CDDP-containing CPT arm. Diarrhoea was more frequent in the CPT-containing arm than in the VDS-P arm. In the present trial, there were no deaths associated with the treatment in the CPT-P arm, but one patient in the CPT arm developed diarrhoea after recovery from myelosuppression and subsequently died from infection. In this patient, it was not clear whether the source of infection was the bowel or the tip of the catheter. In 10 patients treated with the regimen containing CPT-11 who developed grade 3 or higher infection, seven patients developed diarrhoea coincidentally with myelosuppression, which suggested enteritis as the source of infection. Infection was avoided in many patients by taking proactive measures against aggravation of diarrhoea and taking early anti-infection measures.In conclusion, the response rate was significantly higher in patients with CPT-P therapy compared with VDS-P, and the survival time and toxicity were comparable, although this therapy failed to demonstrate significant survival prolongation. Based on these results, a large-scale randomised phase III trial comparing CPT-P as the control regimen with three new drugs  containing platinum-based doublets is currently underway in Japan. CPT-11 monotherapy produced survival result that was not inferior to that obtained with VDS-P, but produced less toxicity. Further studies are necessary to determine the significance of CPT-11 monotherapy in the treatment of NSCLC. This randomised phase III trial has demonstrated that the regimen containing CPT-11 is one of the most active and well tolerated in the treatment of advanced NSCLC.


Cardiac dysfunction is a common complication associated with major burns. While recent findings have linked the Th-17 T-cell response to the development of autoimmune myocarditis, the role of IL-17 and the Th-17 T-cell response in the development of post-burn cardiac dysfunction remains unknown.rd degree, 25% TBSA) or sham treatment. Three hours after injury plasma and tissue  samples were collected and analyzed for the expression of Th-17 cytokine  levels by ELISA.Male C57BL/6 mice were subjected to a major burn  and Th-2 (IL-10) cytokines were not significantly affected after injury. The cardiac Th-17 response correlated with a significant increase in Troponin levels at 3 hr. after burn.These findings indicate that early after burn, cardiac tissue is associated with significantly elevated levels of Th-17 cytokines. The early Th-17 response after burn appears to be specific for cardiac tissue and may promote myocardial inflammation and dysfunction associated with this form of trauma. Burn injury initiates changes in the immune function that result in local and systemic inflammation that can lead to life-threatening end-organ dysfunction. Cardiac dysfunction after burn has been well described and has been shown to play an important role in patient outcome . SeveralRecently a novel class of T-helper cells, called Th-17 cells, has been found to secrete the pro-inflammatory cytokine IL-17 . This suThe importance of cardiomyocyte injury after burn has been emphasized in recent years, however, its pathogenesis, has not been fully clarified. In the current study we examined the role of the Th-17 mediated inflammatory response in the development of the cardiac injury after burn in a mouse model.C57BL/6 male mice  were used for all experiments. The mice were allowed to acclimatize in the animal facility for at least 1 week prior to experimentation. Animals were randomly assigned into either a thermal injury group or a sham treatment group. The experiments in this study were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio, and were performed in accordance with the National Institutes of Health guidelines for the care and handling of laboratory animals.Mice received a scald burn as described previously . BrieflyAt 3 hr after injury the mice were euthanized and tissue samples collected . The tissue samples were snap frozen in liquid nitrogen and stored at -80°C prior to analysis.Tissue samples were homogenized in protease inhibitor cocktail as previously described prior to analysis . Plasma P value of < 0.05 was considered to be statistically significant for all analyses.Data are expressed as mean ± SE. Comparisons were analyzed using ANOVA. A P < 0.05) elevation in the cardiac levels of IL-17, 3 hours after injury as compared to the sham animals. One day after burn, IL-17 remained elevated, however, without significant difference from the respective sham group. Sham levels of IL-17 were significantly (P < 0.05) greater at 3 hrs and 1 day as compared with days 3 and 7. This early cytokine response is likely related to volume loading early after resuscitation, as both sham and burn mice received resuscitation fluid. Based on the observation that the IL-17 response was significantly higher at 3 hrs after burn, all subsequent analysis was conducted on that group of animals.Sham and burn groups were composed of 6 and 5 animals, respectively. There were no animal deaths after burn or sham procedures. At 3 hrs, 1 day, 3 days and 7 days after the burn or sham treatment, animals were euthanized and the heart removed. As shown in figure P < 0.05) levels of IL-17, IL-6 and IL-22 in the burn group as compared to the sham animals. The TGF-β response was not different between groups and IL-23 was virtually undetectable in cardiac tissue samples.In addition to IL-17, we analyzed the heart for the Th-17 family cytokines IL-6, IL-22, IL-23 and TGF-β. As shown in figure We also investigated the effects of burn in other tissues at 3 hrs after injury. Table We found that burn was associated with increased Troponin-I levels at 3 hours after injury Fig. , indicatTo determine whether the cytokine response in the heart after burn was also related to changes in the Th1 and Th2 we analyzed heart tissue for IFN-γ or IL-10 as specific markers of the Th1 and Th2 response respectively. IFN-γ was not detectable irrespectively of the group and IL-10 levels were similar in sham and burned animals at 3 hrs after injury. (Table The inflammatory cascade elicited by burn activates a broad immunoinflammatory response known as the Systemic Inflammatory Response Syndrome (SIRS). This response involves a wide range of cells  and tissues . Ultimately, SIRS increases the potential for the development of Multiple Organ Dysfunction Syndrome (MODS). Since the initial studies of Blalock in the 1930's about cardiovascular impairment after burn, many models have been proposed to explain its pathophysiology including fluid shift, changes in the microcirculation, coagulopathy and peripheral vasoconstriction, however, the most recent research studies have been focused on the association of certain factors with the cardiac dysfunction after burns, including complement, cellular apoptosis and cytokines as the purpose of the current study ,11-14. TStudies by Finnerty et al. have recently shown that circulating IL-17 levels are increased early after burn in pediatric patients, as well in a mouse burn model ,20. ThesEvidence points to IL-17 as the major effector molecule produced by Th-17 cells. IL-17 stimulates several cell types, such as endothelial cells, epithelial cells and macrophages to produce multiple pro-inflammatory mediators, including IL-6 and IL-23 . It alsoInterventions in the complex inflammatory cascade following burn might improve morbidity and mortality in this patient population. These initial findings suggest that IL-17 may provide a unique target of therapeutic intervention to reduce cardiac dysfunction after burn.IL: interleukin; TBSA: total body surface area; TGF-β: Transforming growth factor-Beta; IFN-γ: Interferon gamma; TNF-α: tumor necrosis factor alpha; HMGB-1: High-mobility group box-1; MPO: myleoperoxidase; SIRS: Systemic Inflammatory Response syndrome; MODS: Multiple Organ Dysfunction Syndrome.The authors declare that they have no competing interests.JS and QZ were responsible for the animal experiments and ELISAs. MR was responsible for ELISA analysis, data analysis and scientific interpretation. RO was responsible for the data analysis, scientific interpretation and drafted the manuscript. MS was responsible for scientific conception, design and helped to draft the manuscript. All authors read and approved the final manuscript.


In the rectal carcinomas, there was a significant inverse association between the expression of p120-catenin in cell membranes of the primary tumors and the occurrence of local recurrence, while membranous protein expression of β-catenin was inversely related to distant metastases.The E-cadherin-catenin complex provides cell-cell adhesion. In order for a carcinoma to metastasize, cancer cells must let go of their hold of neighboring cells in the primary tumor. The presence of components of the E-cadherin-catenin complex in 246 rectal adenocarcinomas was examined by immunohistochemistry and compared to their presence in 219 colon carcinomas. The expression data were correlated to clinical information from the patients' records. There were statistically significant differences in protein expression between the rectal and the colon carcinomas regarding membranous  It is located at the cell membrane of epithelial cells and consists of a chain of proteins. The outermost element is E-cadherin, also called uvomorulin, which is a transmembrane glycoprotein . E-cadheto actin . α-caten-catenin , 11 or γkoglobin , which i-catenin  and γ-ca-catenin  also areβ-catenin, that is not bonded, translocates from the cytosol to the nucleus. Here, in a complex with T cell factor (TCF) and Lymphoid enhancer-binding factor (LEF), β-catenin controls expression of several genes, among them Snail1 [β-catenin that would otherwise be combined with E-cadherin in the E-cadherin-catenin complex by the cell membrane [β-catenin can enter the Wingless pathway, so causing increased expression of Snail1.In the Wingless (=Wnt) pathway, m Snail1 . Snail1 m Snail1 . In EMT,m Snail1 , 17. Themembrane . The libβ-catenin signaling, whereas higher levels of Kaiso expression inhibit β-catenin signaling [p120-catenin stabilizes E-cadherin by controlling E-cadherin turnover at the cell membrane. p120 absence dramatically accelerates E-cadherin degradation , 20. p12ignaling .Adherens junctions are cellular structures found near the apical surface of polarized epithelial cells. They provide cell-cell adhesion. The E-cadherin-catenin complex is typically found in adherens junctions , 26, butIn order for a carcinoma to metastasize, cancer cells must detach from neighboring cells in the primary tumor. This process requires a malfunction of the E-cadherin-catenin complex. Several studies have demonstrated reduced expression of E-cadherin –35 and cA simultaneous investigation of the protein expression of both E-cadherin and all the catenins in the E-cadherin-catenin complex in only rectal carcinomas has so far never been performed. Most of the previous studies have included both rectal and colon carcinomas. Since there are clinical differences in prognosis and outcome between patients operated for rectal and colon carcinomas, as well as reports about tumor biological differences between these two tumor types , we aimeAvailable paraffin-embedded tumor samples from a consecutive series of 274 rectal adenocarcinomas removed surgically at Akershus University Hospital in the years 1992–2000 were scrutinized for inclusion into the survey. These surgical treatments were all primary operations. Tumors at a level of 15 centimeters (5.9 inches) or less from the anal verge  were included (246 patients), in accordance with the somewhat arbitrary range of 15 to 18 centimeters commonly used to define the border between rectum and colon. The most restrictive border was chosen in order to avoid unintentional inclusion of sigmoid tumors. The clinico-histopathological characteristics of the patients are shown in Out of 246 patients, 25 experienced a local recurrence of their rectal cancer. Among those included in the statistical analyses, mean time from primary operation to local recurrence was 2.4 years. Minimum time was six months, maximum 6.0 years. For patients with no local recurrence, mean observation time was 6.7 years (range 4 days–14.5 years). Out of 246 patients, 49 developed distant metastases from their rectal cancer. Among those included in the statistical analyses, mean time from primary operation to distant metastases was 2.3 years. Minimum time was two months, maximum 12.9 years. For patients without distant metastases, mean observation time was 7.2 years. Minimum observation time was four days, maximum 14.4 years.This material of rectal carcinomas was compared to a material examined by Bondi et al. of 219 colon carcinomas operated on at Akershus University Hospital during the years 1988, 1990, and 1997–2000 , 50.Serial sections (3-4 micrometers) from formalin fixed, paraffin wax embedded archive tumor tissue were applied to coated slides before immunohistochemical staining, where different methods were used.β-catenin, sections were dried at 50–60°C overnight, deparaffinized and rehydrated before antigen retrieval was performed using microwave technique (20 minutes at 100°C). Staining was done in Dako Autostainer .For α-catenin, p120-catenin, and E-cadherin were dried overnight at 50–60°C before pretreatment with Dako PT link (20 minutes in 98°C), and staining in Dako Autostainer , using Dako's EnVision Flex-system, with mouse-linker in p120-catenin and E-cadherin.Sections for γ-catenin, sections were fixed in incubator 30–40 minutes at 56°C, and overnight at 37°C. The immunostaining was performed in Ventana ES Benchmark automated slide stainer , antibody diluent (251-018). The details of each antibody used are shown in For The antibodies were visualized for light microscopy with Envision Plus-System and diaminobenzidine (DAB), and with Detection Kit Ventana iViewTM DAB, respectively. Counter-staining was done with Hagen's haematoxylin for visualization of tissue structures. Positive control was a test block with normal colon mucosa and multiple colonic adenocarcinomas with diverse differentiation.The percentage of positive cell membranes was counted semiquantitatively by applying four defined grades of immunopositivity. When 60% or more of the cancer cells were stained, we scored the tumor as grade three. Staining of 30%–59% of the cells was classified as grade two. When 5%–29% of the cells were stained, the score was grade one. Positivity in less than five percent of cells qualified for grade zero. The protein expression was regarded positive regardless of whether the expression was mainly at the apical portion of the cell , 52 or iβ-catenin was done in the same way as for the membranes, except for that the border between grade zero and grade one was set to zero percent of the nuclei being stained. The reason for this is that normally, there should be no staining of nuclear β-catenin at all. Only clearly nuclear staining was recorded as positive.Scoring of nuclear staining of We intended to compare our results from rectal cancers to results from a similar examination of colon cancers previously performed. Therefore, our method for quantifying proteins in question had to be the same, with the same cut-offs between the scored grades. Originally, the choice of four levels of staining were applied as a compromise between the wish of detailed measurements and the wish of not giving the impression of being more accurate than manual human judgment is in real life. One intention with the system was that alterations in protein staining in a cancer should be larger than alterations naturally occurring in normal mucosal tissue before they were recorded as pathological. For most slides, we were able to check that this intention was followed, because almost all slides contained normal adjacent mucosa in addition to the cancer. The normal mucosa served as an internal control. In other, earlier, similar investigations, varying cut-offs have been used, and no unified, generally applied standard seems to exist for this. So, in addition to the above mentioned, the original cut-offs were somewhat arbitrarily chosen.At least 100, usually more than 1000 cells, were examined in each slide. All slides were primarily judged by RAA. In addition, judgment of slides was done by AB, IRKB, JB, or SNA, depending on the type of slide in question. When necessary, consensus was achieved by simultaneous examination by two examiners in a double microscope.P < .05 was chosen.Statistical analyses were performed by SPSS version 16.0 running on Windows XP. Binary logistic regression analysis and Cox regression analysis were performed. We made test plots for proportional hazards for the Cox analyses and found them satisfactory. An alpha level of statistical significance of α-catenin, β-catenin, γ-catenin, p120-catenin, and E-cadherin, and also nuclear β-catenin, into the same multivariate analysis.In all statistical analyses, we included patient gender, tumor differentiation grade, Dukes' tumor stage, and patient age at surgery as well as the five membrane proteins Figures β-catenin in the rectal and the colon adenocarcinomas are shown in β-catenin, γ-catenin, p120-catenin and E-cadherin, and regarding nuclear staining of β-catenin . There was also a significant inverse relationship between the expression of β-catenin in cell membranes of the primary tumors of the rectal cancers and the occurrence of metastases . In the rectal carcinomas, there was a significant inverse relationship between the expression of p120-catenin in cell membranes of the primary tumors and the occurrence of local recurrence , a process crucial for the metastatic process. The present study indicates that the EMT process may be different in rectal and colon adenocarcinomas within the same tumor stage.Results from the present study strongly indicate that rectal and colon adenocarcinomas should be separated before analyzing the prognostic value of proteins involved in the adhesion complexes of the cells.The present research is one of the first studies demonstrating tumor biological differences with significance on protein level between rectal and colon adenocarcinomas, regarding the metastatic process. These differences in tumor biology may explain some of the differences in clinical behavior of these two tumor types, but such differences are so far not taken into account when planning the treatment options. The observation that p120-catenin may be related to decreased risk of experiencing local relapse may help stratifying patients, together with other modalities, in risk groups for relapse.


Lactococcus lactis has been well-characterized over the years as a safe microorganism to produce heterologous proteins and to be used as a safe, live vaccine to deliver antigens and cytokines of interest. Here we report a recombinant strain of L. lactis genetically modified to produce and secrete biologically active IP-10.Chemokines are a large group of chemotactic cytokines that regulate and direct migration of leukocytes, activate inflammatory responses, and are involved in many other functions including regulation of tumor development. Interferon-gamma inducible-protein-10 (IP-10) is a member of the C-X-C subfamily of the chemokine family of cytokines. IP-10 specifically chemoattracts activated T lymphocytes, monocytes, and NK cells. IP-10 has been described also as a modulator of other antitumor cytokines. These properties make IP-10 a novel therapeutic molecule for the treatment of chronic and infectious diseases. Currently there are no suitable live biological systems to produce and secrete IP-10. L. lactis expression plasmid under the regulation of the pNis promoter. By fusion to the usp45 secretion signal, IP-10 was addressed out of the cell. Western blot analysis demonstrated that recombinant strains of L. lactis secrete IP-10 into the culture medium. Neither degradation nor incomplete forms of IP-10 were detected in the cell or supernatant fractions of L. lactis. In addition, we demonstrated that the NICE (nisin-controlled gene expression) system was able to express IP-10 "de novo" even two hours after nisin removal. This human IP-10 protein secreted by L. lactis was biological active as demonstrated by Chemotaxis assay over human CD3+T lymphocytes.The IP-10 coding region was isolated from human cDNA and cloned into an L. lactis forming an effective system to produce IP-10. This recombinant IP-10 is biologically active as demonstrated by its ability to chemoattract human CD3+ T lymphocytes. This strain of recombinant L. lactis represents a potentially useful tool to be used as a live vaccine in vivo.Expression and secretion of mature IP-10 was efficiently achieved by  Interferon-gamma inducible-protein-10 (IP-10) is a C-X-C cytokine that belongs to the subfamily of chemokines and is secreted by T cells, monocytes, endothelial cells, and keratinocytes . IP-10 ein vivo -7. Microytogenes . Anotherof IP-10 . Relatedparasite . All theparasite . To dateinterest -16. Thisdiseases ,17,18.L. lactis to produce and secrete chemokine IP-10. This effort is impelled by the solid precedents that have demonstrated that L. lactis is a safe microorganism that could be used for the production of molecules of medical interest, intended to cause immune responses and be employed as a live vaccine.In our work we genetically manipulated the food-grade bacterium E. coli DH5α. The identity of the human ip-10 gene was confirmed by sequencing. This construction was digested with NsiI-EcoRI releasing the IP-10 coding region without ATG and then subcloned into the pVE:3684-E7 vector digested with the same enzymes. This IP-10 coding region was subcloned in-frame with the secretion-signal peptide-encoding sequence of the usp45 gene of L. lactis and under control of the nisin-inducing promoter Pnis [L. lactis NZ9000 by electroporation. Transformants of L. lactis were referred as the NZpSEC:huIP-10 strain. Table To obtain a template to amplify the coding sequence for the mature IP-10, a cDNA was generated from total RNA isolated from human macrophages . A 231-bp DNA fragment encoding IP-10 was amplified and cloned into the pCR 2.1 vector. This new plasmid was named pCR-hIP-10 and was transformed into ter Pnis . This fiL. lactis. In most cases an efficient and correct expression was achieved, but sometimes a poor secretion or intracellular retention of incomplete proteins have been reported, even when this is directed by a secretion-signal peptide. We detected by Western blot a band corresponding to the size of the complete IP-10 protein in the supernatant fraction of induced cultures of L. lactis NZ pSEC:huIP-10. We did not find incomplete or immature forms of IP-10 in any fraction.The ability of strain NZpSEC:huIP-10 to produce and secrete IP-10 was examined by Western Blot using anti-human IP10 antibodies. Protein samples were prepared from cell (C) and supernatant (S) fractions of induced (+) and noninduced (-) NZpSEC:huIP-10 cultures. Supernatant fraction analysis from three recombinant-induced strains (+) showed a clear band corresponding to the size of the human IP-10 protein Figure . No signin vivo, we evaluated the ability of this recombinant strain to secrete IP-10 "de novo" over time after induction by nisin. To answer this, an experiment was made to specifically detect new IP-10 secretion after induction by nisin. Usually the time-course protein-expression experiments reported in the literature are done without removing the protein synthesized and accumulated over time, which may lead to misinterpretation. To avoid this we made the experiment shown in Figure L. lactis were induced with nisin . One hour after nisin induction, cells in B and C were washed and suspended in new nisin-free medium (culture A was kept with nisin throughout the experiment). Two hours later cells in culture B were washed to remove the accumulated protein, suspended in new nisin-free medium and allowed to grow three more hours. A sample was taken for IP-10 detection by Western blot from the three cultures at different times, 1, 3, and 6 hours (counting time 0 as the time nisin was added). Results in Figure Because we wanted to generate a lactococcal vehicle able to produce and secrete the human chemokine IP-10, to deliver IP-10 L. lactis is able to secrete recombinant proteins after inducing the activity of the pNis promoter. This experiment demonstrated that cultures of recombinant L. lactis continue expressing and secreting IP-10 for at least two hours after elimination of nisin from the culture media. For this reason, we would expect to find a similar secretion response from L. lactis after its administration into an animal model, where the inductor would no longer be available but where secretion efficiency would be influenced by the host environment.Our time-correlated evaluation of IP-10 secretion at different times, removing all accumulated protein, avoids misinterpretations and allows pointing out the time that L. lactis, as other bacteria, does not cause glycosylation, we needed to determine if this L. lactis -secreted IP-10 was biologically active.Glycosylation is a common event caused by eukaryotic cells in a variety of proteins. This posttranslational modification has been seen to affect the activity of the protein itself. Because IP-10 has been reported as a glycoprotein [L. lactis were sterilized by filtration (0.22 μm) and added to the lower compartment of the Boyden chemotaxis chambers, with 100 000 IL-2 activated lymphocytes added to the upper wells. After 1 hour of chemotaxis and the counting of cells migrating to the lower side of the membrane (the one that was not exposed to the cells), we were able to demonstrate by direct observation that supernatants from cell-free cultures of recombinant strain NZ pSEC:huIP-10 significantly attracted more T lymphocytes than the controls L. lactis (Wild Type) and PBS, as demonstrated by the greater amount of cells on this underside . Cell-free supernatants from cultures of recombinant side Fig . Our conL. lactis-secreted IP-10 showed a significant chemoattraction to CD3+ human lymphocytes, counting > 5000 CD3+ T lymphocytes  flow cytometer using an anti-CD3 antibody Fig . The amoytes Fig . In contL. lactis to secrete the antitumor chemokine IP-10 (Interferon-gamma inducible-protein-10). Its secretion was highly efficient because no immature or incomplete forms of this chemokine were detected in the cytoplasm or the media. Our results demonstrate that the recombinant strain NZ pSEC:huIP-10 of L. lactis produces and secretes biologically active human interferon-gamma inducible-protein-10 (IP-10) by using the nisin-controlled expression (NICE) system, as demonstrated by chemoattraction of human lymphocytes in a chemotaxis assay  broth at 37°C with vigorous agitation. Lactococcus lactis NZ9000 [E. coli and then transferred to L. lactis by electrotransformation as previously described [E. coli and 10 μg/ml of chloramphenicol for L. lactis.Bacterial strains and plasmids used in this work are listed in Table s NZ9000  was growescribed . Clones Vent DNA Polymerase (New England Biolabs) and RT-PCR  as recommended by the manufacturer using the programmable thermal controller PTC-100 .General procedures for DNA isolations and manipulations were made essentially as described . PCR was2 atmosphere. Macrophages were semipurified by removing nonadherent cells. Macrophages were seeded into 6-well-plates (Costar) at 5 × 106 cells/ml in complete RPMI-1640 medium  stimulated with 20 mg/ml of lipopolysaccharides  and incubated at 37°C in a 5% CO2 atmosphere for 5 hours.Human macrophages were isolated from donor whole blood by centrifugation on Histopaque-1077 (Sigma) according to the manufacturer. The layer containing mononuclear cells was carefully recovered, washed, and cultured for 3 hours in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin solution, and 1% HEPES buffer and then incubated at 37°C in a 5% CO6 cells of LPS-stimulated human macrophages (previously cultured and harvested) was isolated using TRizol reagent (Gibco) according to the manufacturer's instructions. The concentration and integrity of RNA was determined by measuring absorbance at 260 nm and analyzed by formaldehyde-agarose gel electrophoresis. The first strand cDNA was synthesized from 1 μg of total RNA by SuperscriptTM II reverse transcriptase (Gibco) and oligo (dT) 12–18 primer and used to synthesize the second strand.The total RNA from 5 × 10L. lactis (OD600 nm = 0.6–0.8) were induced with 10 ng/ml of nisin (Sigma) for one hour.To allow the expression of huIP-10, cultures of recombinant strains of L. lactis were simultaneously induced with 10 ng/ml of nisin at OD600 nm = 0.4 and their growth was followed for 6 hours under optimum conditions. Culture A was left intact and no removal of medium was made at any time. Cultures B and C, after 1-hour induction, were centrifuged, the cell pellet washed with sterile PBS, and resuspended in the same volume of fresh GM17 medium. Culture B underwent the same wash step at hour 3. Protein extraction for all the 3 cultures was done identically at hour 1, 3, and 6.For long-term expression experiments, three cultures of recombinant g at 4°C for 5 minutes and resuspended in 100 μl of TES-lysis buffer  and 10 mM of dithiothreitol (DTT). The mixture was incubated at 37°C for 1 hour and then 50 μl of 20% SDS and one volume of loading buffer were added. The samples were maintained at -20°C before loading onto the gel.Cell and supernatant fractions were prepared separately. Samples were processed from 1.35 ml of culture. Cell pellets were obtained by centrifugation at 21000 × g at 4°C for 15 minutes. The pellet was resuspended in 50 μl of 50 mM NaOH and 50 μl of SDS-PAGE loading buffer . Twenty μl of these preparations were loaded onto 15% acrylamide gels. SDS-PAGE and Western blotting was done essentially as described [The supernatant samples were treated with 1 mM of PMSF and 10 mM DTT to avoid proteolysis. Proteins were precipitated using 150 μl of 100% trichloroacetic acid (TCA) and incubated on ice for 10 minutes followed by centrifugation at 21000 × escribed . Inmunod2 atmosphere for 12 days. Cell density was kept all the time between 1 and 3 × 106 cells/ml.Human-peripheral blood lymphocytes were isolated from donor whole blood by centrifugation on Histopaque-1077 (Sigma) according to manufacturer's instructions. The mononuclear cell layer was carefully recovered and washed three times with RPMI 1640 medium (Gibco). Adherent cells were discarded and cells in suspension were cultured in RPMI 1640 medium supplemented with 5% human serum (Sigma) and 200 U/ml human IL-2 (Santa Cruz Biotechnologies) at 37°C in a 5% COL. lactis, sterilized by filtration through a 0.22-μm filter , were loaded into the lower compartment of the chamber. Lymphocytes (1 × 105 cells) in RPMI 1640 medium were loaded into the upper compartment of the chamber. Chemotaxis was allowed to occur for 1 hour at 37°C in 5% CO2. The membrane from the chamber was removed, washed with PBS, fixed, and stained with hematoxylin. Cells that had migrated to the underside of the filter were fixed with methanol and stained with hematoxylin. The number of migrated cells was counted by using light microscopy. For each membrane, five randomly selected fields were counted. Cells migrating into the lower compartment of the chamber were counted by a fluorescence-activated cell-sorting (FACS) flow cytometer . For this flow cytometry assay, the aqueous phase from the lower chambers was gently recovered and centrifuged. The resulting cell pellet was suspended in PBS and incubated for 10 min with human anti-CD3 FITC , washed with PBS, and counted with the FACS sorting cytometer. All chemotaxis assays were done in triplicate. Complement-activated serum was prepared from fresh human serum by addition of 25 mg of zymosan (Sigma) per ml of serum and incubated at 37°C for one hour. Statistical analysis was done using the Tukey test.Cell migration was measured in 5.0-μm pore-size cellulose-nitrate membranes (Whatman) using a Boyden chemotaxis chamber. Supernatants from recombinant and wild-type The authors declare that they have no competing interests.JVH performed all experiments and drafted the manuscript. MJLA, AGE, MFM, JGGG and HRR participated in all experiments and technically supported the experimental process. YGP, OSC, JVF and RML contributed ideas for performing the experiments, professional support, and helpful suggestions for improving the manuscript. All authors read and approved the final manuscript.


TRPM8 is a non-selective cation channel that belongs to the melastatin subfamily of the transient receptor potential (TRP) ion channels. TRPM8 is activated by voltage, cold and cooling compounds such as menthol. Despite its essential role for cold temperature sensing in mammals, the pharmacology of TRPM8 is still in its infancy. Recently, tyrosine 745 (Y745) was identified as a critical residue for menthol sensitivity of the channel. In this report, we study the effect of mutating this residue on the action of several known TRPM8 antagonists: BCTC, capsazepine, SKF96365, and clotrimazole as well as two new inhibitor candidates, econazole and imidazole.We show that Y745 at the menthol binding site is critical for inhibition mediated by SKF96365 of cold- and voltage-activated TRPM8 currents. In contrast, the inhibition by other antagonists was unaffected by the mutation (BCTC) or only partially reduced , suggesting that additional binding sites exist on the TRPM8 channel from where the inhibitors exert their negative modulation. Indeed, a molecular docking model implies that menthol and SKF96365 interact readily with Y745, while BCTC is unable to bind to this residue.In summary, we identify structural elements on the TRPM8 channel that are critical for the action of channel antagonists, providing valuable information for the future design of new, specific modulator compounds. ACD/ChemSketch Freeware, version 8.0  was used to draw the structures of the various modulators shown in Figure The chemical substances studied for their modulatory effect on the TRPM8 constructs were the cooling agent Experiments were always performed in parallel with cells expressing TRPM8-wt and TRPM8-Y745H to minimize any day-to-day variability in experimental conditions. To estimate the shifts in the voltage-dependence of activation of TRPM8-wt/Y745H in HEK293 cells, current-voltage (I-V) relationships from voltage ramps were fitted with an equation that combines a linear conductance multiplied by a Boltzmann activation term :g is the maximum whole-cell conductance, Erev is the reversal potential, V1/2 is the potential for half maximal activation and s is the slope factor. For each cell, fitting was started by analyzing a condition exhibiting inward current. The value obtained for the parameter Erev at this condition was used when fitting I-V curves that lacked inward current.where http://pubchem.ncbi.nlm.nih.gov/ and used with no additional optimization. The protein-ligand docking and the analysis of interactions was accomplished with Autodock [http://www.pymol.org).TRPM8-ligand complexes were built using the TRPM8 model constructed by Pedretti et al. . Ligand Autodock  implemenAutodock , and optAutodock . DockingA TRPM8 antibody was generated in the laboratory. Briefly, whole serum anti-TRPM8 was obtained by rabbit immunization with a peptide corresponding to amino acids 1-10 of mouse TRPM8 (MSFEGARLSM) conjugated to keyhole limpet hemocyanin. For immunoblot analysis of TRPM8-wt/Y745H transfected HEK293 cells, 48-h post transfection, cells were mixed with sample buffer (4% SDS (w/v); 10% glycerol (v/v); 4% 2-mercaptoethanol; 0.2% bromophenol blue (w/v); 50 mM Tris-HCl; pH 6.8), heated at 95°C for 5 min, and then subjected to SDS-PAGE gel electrophoresis (10% acrylamide gel). Proteins were transferred onto a nitro-cellulose membrane Hybond™ ECL (Amersham Biosciences). Membranes were blocked with 10% non-fat milk in TBS , and subsequently incubated with the primary antibody for TRPM8 at 1:500 dilution for 1 h at room temperature and at 4°C overnight. Blots were treated with peroxidase-conjugated goat anti-rabbit IgG at 1:2000 dilution for 1 h, and protein signals were revealed using the ECL Advance™ Western Blotting Detection Kit (Amersham Biosciences) and visualized with the LAS-1000 imaging system by Fujifilm.t-test. For multiple comparisons of means, one-way or two-way ANOVA was performed using GraphPad Prism version 4.00 for Windows . Paired or repeated-measures tests were used where appropriate to account for inter-individual variability.Data are reported as mean ± standard error of the mean. All fitting was carried out with the Levenberg-Marquardt method implemented in the Origin 7.0 software. In the dose-inhibition curves shown in Figure The authors declare that they have no competing interests.AM designed and carried out the electrophysiological studies, the statistical analyses, and drafted the manuscript. MP generated the point-mutant TRPM8-Y745H; conducted the calcium imaging studies and their analysis; as well as the immunoassays. GFB carried out the molecular modelling. AFM participated in the design of the molecular modelling and helped to draft the manuscript. FV conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Suppressive immune cells present in tumour microenvironments are known to augment tumour growth and hamper efficacy of antitumour therapies. The amino-bisphosphonate Zoledronic acid (ZA) is considered as an antitumour agent, as recent studies showed that ZA prolongs disease-free survival in cancer patients. The exact mechanism is a topic of debate; it has been suggested that ZA targets tumour-associated macrophages (TAMs).in vivo and in vitro. Mice were intraperitoneally inoculated with a lethal dose of mesothelioma tumour cells and treated with ZA to determine the effects on myeloid differentiation and survival.We investigate the role of ZA on the myeloid differentiation to TAMs in murine mesothelioma We show that ZA impaired myeloid differentiation. Inhibition of myeloid differentiation led to a reduction in TAMs, but the number of immature myeloid cells with myeloid-derived suppressor cell (MDSC) characteristics was increased. In addition, ZA affects the phenotype of macrophages leading to reduced level of TAM-associated cytokines in the tumour microenvironment. No improvement of survival was observed.We conclude that ZA leads to a reduction in macrophages and impairs polarisation towards an M2 phenotype, but this was associated with an increase in the number of immature myeloid cells, which might diminish the effects of ZA on survival. Recently, cancer-related inflammation was recognised as the seventh hallmark of cancer .highGr-1low-MDSC , whereas classically activated macrophages (M1) are more associated with inflammation. These two types of macrophages differ in receptor expression, effector function, and cytokine and chemokine productions .The recruitment of immature myeloid cells is enhanced by the production of chemokine (C–C motif) ligand-2/monocyte-chemotactic protein-1 (CCL-2/MCP-1), which is produced and expressed by TAMs and tumour cells. Within the heterogeneous group of immature myeloid cells, subpopulations are defined, which can further differentiate into mature macrophages, and hence TAMs provide their own precursor cells, which enhances tumour progression  , on TAM formation and tumour progression in a murine mesothelioma model.in vitro and in vivo. In addition, we determined whether ZA changed the phenotype of macrophages. Animal studies were performed to investigate the effect of ZA on survival.In this study, we analysed the effect of ZA on myeloid differentiation both CBA-j mice  were purchased from Harlan  and were housed under pathogen-free conditions at the Erasmus MC animal facility. All experiments were approved by the local ethical committee for animal welfare  and complied with the guidelines for the welfare of animals in experimental neoplasia by the United Kingdom Coordinating Committee on Cancer Research (UKCCCR) and by the Code of Practice of the Dutch Veterinarian Inspection. The AC29 cell line is kindly provided by Professor BWS Robinson .Macrophages were depleted by intraperitoneal (i.p.) injection of liposome-encapsulated clodronate  .6 AC29 tumour cells on day 0. Mice were i.p. injected with 200 μl liposome-encapsulated clodronate or liposome-encapsulated PBS at days 5 and 10 after tumour injection. At day 12 mice were killed using CO2. The peritoneal cavity of tumour-bearing mice was washed with 1 ml PBS to obtain peritoneal cells for FACS analysis. All visible tumour material was excised from each mouse and data are expressed as wet weight (accuracy of 0.001 g).CBA-j mice were i.p. inoculated with a lethal dose of 20 × 10Tumour-associated macrophages were defined by the expression of F4/80, MHCII and CD206 (marker for M2 macrophage phenotype).μm) were cut in an HM-560 cryostat . F4/80  and CD206  primary antibodies were incubated for 1 h at room temperature. Binding of antibodies was detected using the immunoalkaline phosphatase antialkaline phosphatase method . Naphthol-AS-MX-phosphate  and new fuchsine  were used as substrate. The specificity of the antibodies was checked using a protein concentration-matched non-relevant monoclonal antibody and PBS.Tumour material was obtained 25 days after tumour injection from the peritoneal cavity of tumour-bearing mice. Tumour biopsies were embedded in Tissue-Tek II optimum cutting temperature medium , snap frozen in liquid nitrogen and stored at −80°C. Tissue sections , depleted of erythrocytes by osmotic shock, washed twice in RPMI and adjusted to a concentration of 1 × 106 cells ml−1 in FACS buffer.Spleens were aseptically removed and mechanically dispersed in cold PBS. Cell suspensions were filtered through a 100-Splenocytes were stained with the following monoclonal antibodies: Ly6C (FITC), F4/80 (FITC), MHCII (PE), CD11c (PE-Texas red), CD11b (PerCP-Cy5.5), CD31 (PE-Cy7), CD206 (Alexa 647), Ly6G (Alexa Fluor 700), Gr-1 (APC-Cy7) and a fixable live/dead marker in DAPI . The final analysis and graphical output were performed using FlowJo software .−1)  and β-mercaptoethanol (Sigma-Alderich) (further referred to as culture medium).Single-cell suspensions of bone marrow, isolated from femurs and tibias of naïve mice, were cultured in RPMI supplemented with 10% foetal calf serum, 2.5 ml gentamicin  was used in our bone marrow cultures according to the protocol of 6 bone marrow-derived cells. AC29 was cultured in RPMI-1640  supplemented with GlutaMax, 10 mM HEPES and 5% heat-inactivated foetal bovine serum. When confluency reached 80% , supernatant was collected, centrifuged for 10 min at 1000 g to remove cells/cell debris. Supernatant is added to the bone marrow culture at a concentration of 30%. This concentration was selected based on findings by μM. All culture experiments have been repeated five times under comparable conditions.Bone marrow cells were isolated from the femurs and tibias of naïve mice under sterile conditions (Mononuclear-MDSCs were isolated from the spleen of tumour-bearing mice. Four to six colour samples were sorted using a FACSAria equipped with FACSDIVA software (BD Biosciences). After sorting, cells were cultured with M-CSF for 6 days. This experiment has been repeated twice under comparable conditions.6 AC29 tumour cells. One group was treated daily with subcutaneous (s.c.) ZA injections  in 100 μl PBS and the other group was treated with 100 μl PBS as a control starting at day 5. This dosage was proven effective and non-toxic in mice  and VEGF levels in the effusion fluid were determined using a specific ELISA assay : BD Biosciences; VEGF: Calbiochem, Darmstadt, Germany). Protocols were followed as per the manufacturer's instructions. Samples were diluted appropriately to ensure that readings were within the limits of accurate detection. Results are expressed as pg ml−1 of effusion fluid.To measure cytokine levels, effusion fluid from the peritoneal cavity of tumour-bearing mice was obtained at day 25 after tumour injection. IL-10, IL-12, TNFt-tests. A two-tailed P-value <0.05 was considered significant. Data presented as a percentage of tumour-free animals were analysed with Kaplan–Meier survival curves, using the log-rank test to determine significance.Data are expressed as mean±s.d. Comparisons between groups were made using We investigated the effect of the depletion of macrophages on tumour progression in a murine model for mesothelioma by treating mice with liposome-encapsulated clodronate. These liposomes are readily taken up by phagocytic cells, including macrophages, and induce cell-specific apoptosis after clodronate is set free into the cytoplasm of cells .Mice were i.p. inoculated with a lethal dose of AC29 tumour cells and mice were treated with liposome-encapsulated clodronate or liposome-encapsulated PBS on days 5 and 10 after tumour injection. Mice were killed 12 days after tumour injection. All visible tumour material was excised from each mouse and wet weight was measured . All mice (n=5) treated with liposome-encapsulated PBS showed profound tumour growth at day 12. Three of the five mice treated with liposome-encapsulated clodronate had no visible tumour. In the case of mice that did develop tumour, tumour growth was less profound . The expression of Gr-1 remained high in both these culture conditions  of these markers on day 6 of culture (nditions .P<0.0001)  .in vitro, leading to a significant reduction in the MFI of CD206 on macrophages cultured in the presence of ZA (P<0.0001).In conclusion, these data showed that the addition of ZA to macrophage-inducing culture conditions significantly inhibits the upregulation of F4/80, MHCII and CD11c. In addition, these data reveal that addition of tumour supernatant leads to polarisation of the macrophage phenotype towards M2, and that ZA can prevent this polarisation Next, we investigated whether treatment with ZA had an effect on survival. Mice were i.p. inoculated with AC29 tumour cells and treated daily with s.c. ZA injections or PBS as a control. The dosing schedule was chosen according to the literature, in which it was proven effective and non-toxic (P=0.2183)   . No signctively) . Long-tectively) .Taken together, although no significant differences on tumour progression and survival could be observed between untreated mice and mice treated with ZA, a reduction in the number of macrophages and an increase in the number of immature myeloid cells was detected.highGr-1high MDSC (PMN-MDSC), mononuclear CD11bhighGr-1int MDSC (MO-MDSC) and the CD11bhighGr-1low-MDSC  or PBS as a control. Mice were killed 25 days after tumour injection.As we observed a difference in the number of macrophages and immature myeloid cells during ZA treatment, we next wanted to know whether ZA treatment also affects macrophage phenotype +Gr-1+ cells was found in the spleen and effusion fluid of tumour-bearing mice of both groups. A significant increase was found in MO-MDSC both in the spleen and in the effusion fluid from the peritoneal cavity , whereas no significant differences were found in the proportion of PMN-MDSC and Gr-1low-MDSC . The proportion of macrophages in the spleen of ZA-treated mice was significantly lower compared with PBS-treated mice (P=0.0091). There was a trend towards a shift in macrophage phenotype from M2 to M1; however, the reduction in M2 macrophages was not significant (P=0.18). However, we found that the expression levels of CD206, expressed as the MFI, were significantly lower on M2 macrophages of ZA-treated mice compared with M2 macrophages in the spleen of PBS-treated mice (P=0.0095) (P=0.75). However, a significant increase in M1 macrophages was found, while M2 macrophages were reduced (P=0.035 and 0.33). A shift from M2 macrophage to M1 macrophage phenotype was found based on the MFI expression of CD206, confirming the findings in the spleen (P=0.14). As a result, a difference in ratio of M1:M2 was found (P=0.011) (An increase in CD11b=0.0095) . No signP=0.011) .β in the effusion fluid of ZA-treated mice . In addition, we observed a significant reduction in VEGF and CCL-2 (MCP-1) in the effusion fluid of ZA-treated animals (P=0.05 and 0.039). Levels of IL-10 and TNFα were not detectable in the effusion fluid.Enzyme-linked immunosorbent assay on the effusion fluid was performed to determine whether the reduction on CD206 expression on macrophages in ZA-treated mice was accompanied by changes in cytokine, chemokine and growth factor production. A significant increase was found in the levels of IL-6, IL-12 and IL-1β  levels and a significant increase in the levels of IL-6, IL-12 and IL-1β .Many effects of bisphosphonates have been reported in literature. Treatment with ZA prolongs the 1-year survival rate in breast cancer, prostate cancer and bladder cancer  was found on macrophages in tumour-bearing mice treated with ZA. Changes in M2-associated cytokines, chemokines and growth factors were observed in the effusion fluid after ZA treatment. We showed that ZA lowered the levels of VEGF in the effusion fluid of tumour-bearing mice. The reduction in VEGF and MMP-9 expressions by macrophages under the influence of ZA has been described to prevent tumour neovascularisation  due to treatment with ZA. Recently, studies have reported the role of MDSC in tumour progression. MDSC are most known for their immune suppressive function and induction of tumour-specific T-cell tolerance facilitating tumour immune escape . Therefoet al. reported similar observation. In their study, repolarisation of the macrophage phenotype was most promising during early tumour development, resulting in prolonged tumour-free survival and overall survival. In addition, they notify that shifting the phenotype in a host with a large tumour burden can be hazardous and no effects of ZA administration were observed when in situ carcinomas had progressed. From their data, it appears that tumour development is inhibited when the induction of M2 macrophages can be post-poned. We observed similar findings: shifting the phenotype from M2 to M1 at later stages of tumourigenesis seems less effective. Therefore, it can be hypothesised that preventing the formation of M2 macrophages is a critical key in preventing tumour outgrowth during early stages of disease development.A second reason as to why ZA has no effect on survival may be assigned to the shift in macrophage phenotype. As most studies showed that TAMs are polarised to an M2 phenotype that contribute to carcinogenesis, shifting the phenotype of macrophages to M1 seems promising. M1 macrophages produce less tumour growth-promoting soluble factors and can potentially kill tumour cells. However, the question rises whether shifting the phenotype in full-blown tumours is possible and whether repolarisation is as promising as believed previously . NegativA third explanation for the limited effect on survival could be that ZA has effects on processes during tumour development that do not have a dominant role in our tumour models. Most investigations are carried out in tumours with high metastasis rates in combination with other therapeutic approaches (like adjuvant endocrine therapy) . In thesN-telopeptide/creatinine ratio) and preliminary evidence of efficacy on skeletal-related events; however, no studies were performed comparing dose–effect relations on the tumour microenvironment. Although there are some concerns on side effects caused by ZA at higher levels (To study the effects of ZA on macrophages in the primary tumour, dose levels are higher than clinically advised. Doses selected for clinical trials were based on changes in bone resorption markers (including serum COOH-terminal telopeptide and urinary In conclusion, our data show that daily administration of ZA in tumour-bearing mice inhibits myeloid differentiation and shifts the balance from M2 macrophage phenotype to M1 macrophage phenotype. However, as a consequence the number of immature myeloid cells remains high in the spleen and in the effusion fluid, especially in the MO-MDSC population. This MO-MDSC population was found to have an immunosuppressive effect to which we ascribe the fact that survival was not improved with ZA in our study.